A Sparse Matrix Screen to Establish Initial Conditions for Protein Renaturation

Hofmann, Adele D.; Tai, Mei-Sheng; Wong, Wan Keung; Glabe, Charles G.

doi:10.1006/abio.1995.1429

Cited by 14 publications

(8 citation statements)

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“…Although important, pH and reductants are not the only variables to consider when designing a refolding screen. Studies have shown that a single protein can refold under markedly different conditions (Hofmann et al 1995; Armstrong et al 1999). Our data set contained two phosphatases with 65% sequence identity and nearly identical structural folds.…”

Section: Discussionmentioning

confidence: 99%

“…In practice, however, there is no universal method or buffer for reliably refolding a given protein of interest and identification of initial refolding conditions remains a major hurdle. One way to overcome this obstacle is by the introduction of refolding screens to rapidly identify initial conditions that result in folded protein (Hofmann et al 1995;Chen and Gouaux 1997;Armstrong et al 1999;Tobbell et al 2002;Maxwell et al 2003;Scheich et al 2004;Tresaugues et al 2004;Vincentelli et al 2004). These screens were designed to test a variety of refolding additives in a minimal number of experiments.…”

Section: Discussionmentioning

confidence: 99%

“…Fractional factorial refolding screens have emerged as a way to compensate for this unpredictability. Fractional factorial screens contain a representative subset of reagent combinations contained in full factorial screens and are designed to maximize the number of refolding variables explored while minimizing the amount of data collection (Hofmann et al 1995; Chen and Gouaux 1997; Armstrong et al 1999; Tobbell et al 2002). These screens have been used successfully to refold proteins, but the choice of refolding additives included in these screens is based on historical precedent and does not take into account novel reagents shown to improve protein renaturation.…”

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confidence: 99%

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Investigation of protein refolding using a fractional factorial screen: A study of reagent effects and interactions

et al. 2005

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A recurring obstacle for structural genomics is the expression of insoluble, aggregated proteins. In these cases, the use of alternative salvage strategies, like in vitro refolding, is hindered by the lack of a universal refolding method. To overcome this obstacle, fractional factorial screens have been introduced as a systematic and rapid method to identify refolding conditions. However, methodical analyses of the effectiveness of refolding reagents on large sets of proteins remain limited. In this study, we address this void by designing a fractional factorial screen to rapidly explore the effect of 14 different reagents on the refolding of 33 structurally and functionally diverse proteins. The refolding data was analyzed using statistical methods to determine the effect of each refolding additive. The screen has been miniaturized for automation resulting in reduced protein requirements and increased throughput. Our results show that the choice of pH and reducing agent had the largest impact on protein refolding. Bis-mercaptoacetamide cyclohexane (BMC) and tris(2-carboxyethylphosphine) (TCEP) were superior reductants when compared to others in the screen. BMC was particularly effective in refolding disulfide-containing proteins, while TCEP was better for nondisulfide-containing proteins. From the screen, we successfully identified a positive synergistic interaction between nondetergent sulfobetaine 201 (NDSB 201) and BMC on Cdc25A refolding. The soluble protein resulting from this interaction crystallized and yielded a 2.2 Å structure. Our method, which combines a fractional factorial screen with statistical analysis of the data, provides a powerful approach for the identification of optimal refolding reagents in a general refolding screen. Keywords: protein folding; fractional factorial screen; crystal structure; inclusion bodies; highthroughput refolding; structural genomicsThe identification of 20,000-25,000 genes from the human genome project has resulted in a wealth of potential targets for structural biology investigation and pharmaceutical design (International Human Genome Sequencing Consortium 2004). Since the completion of the project, expectations have been high that the number of protein crystal structures would dramatically increase but, in reality, there has only been a moderate rise in the number of crystal structures, due largely to a lack of sufficient quantities of protein suitable for structural studies (Service 2002). Although the technology responsible for expressing recombinant proteins is highly developed , it is still difficult to produce enough soluble protein for these structural studies. The ultimate goal of determining crystal structures on a genome-wide scale requires methods designed to improve the yield of functional protein.Reprint requests to: Ted Fox, Vertex Pharmaceuticals, 130 Waverly Street, Cambridge, MA 02139, USA; e-mail: ted_fox@vrtx.com; fax: (617) 444-6820.Abbreviations: AMP-PNP, 5 0 -adenylylimidodiphosphate; bME, b-mercaptoethanol; BMC, bis-mercaptoacetamide...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Investigation of protein refolding using a fractional factorial screen: A study of reagent effects and interactions

et al. 2005

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“…Aggregation and precipitation of protein during refolding are commonly encountered problems. Factorial and other screens for refolding have been developed (Hofmann et al 1995;Armstrong et al 1999;Bajaj et al 2004) to facilitate the search of appropriate refolding conditions. In the present study we describe the first application of the technique of three-phase partitioning (TPP) (Lovrien et al 1995;Dennison and Lovrien 1997;Jain et al 2004;Przybycien et al 2004) to obtain active refolded proteins from solubilized inclusion bodies without any chromatographic steps.…”

Section: Introductionmentioning

confidence: 99%

Refolding and simultaneous purification by three‐phase partitioning of recombinant proteins from inclusion bodies

et al. 2008

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Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of threephase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP-refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies.Keywords: three-phase partitioning; protein refolding; recombinant ribonuclease A; CcdB mutants; human CD4; inclusion bodies Supplemental material: see www.proteinscience.org Upon expression in Escherichia coli, many recombinant proteins form dense, insoluble aggregates called inclusion bodies (Taylor et al. 1986). It is therefore necessary to develop efficient and scalable procedures for solubilization and refolding of recombinant proteins from inclusion bodies (Misawa and Kumagai 1999;Middelberg 2002).Refolding is typically carried out using either simple dilution into appropriate refolding conditions, dialysis or on a column (Stempfer et al. 1996;Rogl et al. 1998). Finding appropriate refolding conditions is generally the most difficult step of the purification process. Aggregation and precipitation of protein during refolding are commonly encountered problems. Factorial and other screens for refolding have been developed (Hofmann et al. 1995;Armstrong et al. 1999;Bajaj et al. 2004) to facilitate the search of appropriate refolding conditions. In the present study we describe the first application of the technique of three-phase partitioning (TPP) (Lovrien et al. 1995;Dennison and Lovrien 1997;Jain et al. 2004;Przybycien et al. 2004) to obtain active refolded proteins from solubilized inclusion bodies without any chromatographic steps. We have used TPP to refold 12 different proteins from inclusion bodies, namely, ribonuclease A Reprint requests to: Munishwar N. Gupta, Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India; e-mail: munishwar48@yahoo.co.uk; fax: 91-011-26581073.Abbreviations: CcdB, controller of cell division or death B; CD4D12, first two domains of human CD4; C m , the denaturant concentration at which one half of the protein molecules are unfolded; GdmCl, guanidium chloride; IPTG, isopropyl b-D-1-thiogalactopyranoside; MBP, maltose binding protein; PTPs, protein tyrosine phosphatases; RNase A, ribonuclease A; SPR, surface plasmon resonance; TPP, three-phase partitioning; Trx, E. coli thioredoxin.Article and publication are at

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“…8,9,30 A large series of low molecular weight additives are, in certain cases, very efficient refolding enhancers: for examples, nondenaturing concentrations of chaotrophs such as urea or Gdn-HCl are essential for the renaturation of reduced chymotrypsinogen A. 31 The most popular additive is L-arginine.…”

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confidence: 99%

Refolding of therapeutic proteins produced inEscherichia coli as inclusion bodies

1999

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Overexpression of cloned or synthetic genes in Escherichia coli often results in the formation of insoluble protein inclusion bodies. Within the last decade, specific methods and strategies have been developed for preparing active recombinant proteins from these inclusion bodies. Usually, the inclusion bodies can be separated easily from other cell components by centrifugation, solubilized by denaturants such as guanidine hydrochloride (Gdn‐HCl) or urea, and then renatured through a refolding process such as dilution or dialysis. Recent improvements in renaturation procedures have included the inhibition of aggregation during refolding by application of low molecular weight additives and matrix‐bound renaturation. These methods have made it possible to obtain high yields of biologically active proteins by taking into account process parameters such as protein concentration, redox conditions, temperature, pH, and ionic strength. © 1999 John Wiley & Sons, Inc. Biopoly 51: 297–307, 1999

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A Sparse Matrix Screen to Establish Initial Conditions for Protein Renaturation

Cited by 14 publications

References 0 publications

Investigation of protein refolding using a fractional factorial screen: A study of reagent effects and interactions

Investigation of protein refolding using a fractional factorial screen: A study of reagent effects and interactions

Refolding and simultaneous purification by three‐phase partitioning of recombinant proteins from inclusion bodies

Refolding of therapeutic proteins produced inEscherichia coli as inclusion bodies

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